Sickle cell anemia, the world's most common molecular disease, is the result of a single amino acid substitution at a surface position on the beta chain of hemoglobin. The only difference in primary chemical structure between normal hemoglobin (HbA) and sickle cell hemoglobin (HbS) is the substitution of valine for glutamic acid at the sixth amino acid from the NH.sub.2 -terminal of the beta chain.
The treatment of sickle cell anemia by inhibition of sickling using techniques of protein engineering is known. In particular, it is known that cyanate is useful to prevent the sickling of red blood cells of sickle cell anemia patients. See, for example, U.S. Pat. No. 3,833,724 to Cerami et al. It is also known that isocyanic acid, the reactive form of cyanate will react irreversibly with free amino groups of hemoglobin, primarily at the terminal valine position to decrease the polymerization of deoxyhemoglobin S molecules.
In HbS, the resulting carbamylation of the amino termini of the .alpha.- and .beta.-chains that are present produces definite functional alterations such as an increase in oxygen affinity and a reduction in the Bohr effect. It is believed that carbamylation of the amino termini removes some of the salt bridges that stabilize the deoxy configuration of the hemoglobin tetramer present. Thus, carbamylation of HbS amino termini provides species of molecules which may not participate in the formation of tactoids. It has also been demonstrated that carbamylation of HbS prolongs considerably the survival of the red cells of the HbS homozygocytes without considerably affecting the red cell metabolism.
There are two major problem areas related to the use of cyanate in patients, however. The first problem area involves toxic effects due to non-specific carbamylation. Although the findings from animal experimentation appear rather optimistic, it is known that cyanate is a very reactive chemical with no specific affinity for hemoglobin. Intravenous or intraperitoneal administration of cyanate in mice has been shown to produce carbamylation of several enzymes in tissues other than blood, including the brain. Similar effects have been observed in Macacca Nemestrina after chronic administration of cyanate. Although the functional significance of this non-specific carbamylation remains to be assessed, such findings point to the need for maximum care, particularly in the chronic intravenous use of this drug.
The second problem area involves achieving effective levels of hemoglobin modification in vivo. From the available in vitro evidence, it appears that protection from sickling requires the carbamylation of at least one amino terminal valine per HbS tetramer. Such levels of carbamylation are not easily achieved with oral administration of non-toxic doses of cyanate; the reported carbamylation levels in homozygous sicklers treated with cyanate by mouth have been about 0.3 carbamyl groups per tetramer. This low degree of carbamylation may explain the relatively unimpressive effects in patients routinely treated with cyanate orally. An effective degree of carbamylation with intravenous administration of cyanate in an attempt to affect a sickling crisis is out of the question because toxic doses of the drug may be reached before one achieves the desired therapeutic effects. On the other hand, the intravenous administration of 5 to 10 gm of cyanate (LD.sub.50 =250 mg/kg) to sickle cell anemia patients, has resulted in only 0.4 to 0.6 carbamyl groups per tetramer.
To overcome the foregoing problems, extracorporeal treatment of whole blood with cyanate has been suggested, followed by removal of unreacted cyanate from the treated blood by hemodialysis before the blood is returned to the patient. In this manner, efficient carbamylation of HbS can be effected within a relatively short time period. However, in view of the recognized toxicity of cyanate, it is necessary to have a rapid, effective means for detecting the cyanate concentration in blood after hemodialysis so that the efficacy of cyanate removal during the hemodialysis step can be monitored and also so that free cyanate in the blood returned to the patient does not exceed a physiologically tolerable level.
To this end, it has been proposed to introduce cyanate into the blood to be treated as potassium cyanate (KNCO) and to monitor the cyanate concentration in whole blood by a pair of K.sup.+ -sensitive electrodes that compare the concentration of K.sup.+ in blood before and after the treatment, Kjellstrand et al., Trans. Amer. Soc. Artif. Int. Organs, vol. XX, 574-577 (1974). However, the foregoing approach is not satisfactory because the measurement of K.sup.+ concentration in whole blood does not give a reliable indication of the cyanate concentration that may be present because relatively large stores of potassium are already present in the patient's body.
Accordingly, there exists a pressing need for a safe and reliable means for determining the concentration of the cyanate content of whole blood before a practical treatment can be provided to patients suffering from sickle cell anemia. The present invention satisfies this need.